PROJECT SUMMARY/ABSTRACT Our long-term goal in this project is to elucidate the mechanisms of human natural killer cell development in order to apply the processes of NK cell biology in the treatment of cancer. Natural killer (NK) cells recognize malignant cells lacking self-MHC molecules on their surface, thus targeting them for perforin-mediated cytotoxicity. NK cells comprise an important role in the innate immune response to malignancy, and indeed cancers such as acute myeloid leukemia (AML) have been shown to have impaired NK cell development and function. Therefore, understanding the yet unknown mechanisms regulating NK cell maturation in both the normal and disease settings will improve translational research efforts for cancer therapy. Our ongoing work investigates NK cell developmental pathways occurring in secondary lymphoid tissues (SLTs; including tonsils and lymph nodes). In our current studies, we observed that activation of the Notch signaling pathway results in a key transition during development from a non-functional phenotype to a functionally mature NK cell. Moreover, we detected constitutive expression of the transcriptionally-active domain of Notch in mature NK cells freshly isolated from tonsils, thus supporting the physiologic importance of Notch in regulating the functional phenotype of mature NK cells. We hypothesize that Notch signaling in the SLT microenvironment regulates the developmental transition from immature to functional NK cells. We further hypothesize that signaling through the aryl hydrocarbon receptor (AHR), which we observe is activated in AML, alters Notch signaling to inhibit NK cell development. Thus our aims are 1) to determine the mechanism by which Notch is activated in SLTs to promote NK cell functional maturation; and 2) to determine how AHR modulates Notch to antagonize NK cell maturation. To test this hypothesis, we will perform in vitro culture studies and in vivo adoptive transfer experiments using NK and other SLT-resident cells isolated from human tonsils. We will identify the direct gene targets of activated Notch in NK cells to determine how Notch regulates the transcription of critical molecules for NK cell function. Finally, we will identify how AHR modulates Notch signaling in NK cells. The overall significance of this project will address a relevant gap in knowledge regarding the regulation of human NK cell maturation in the SLT microenvironment. This project represents a novel and innovative approach to cancer immunology because it combines both mechanistic studies of an important signaling pathway, and also seeks to identify how cancers such as AML can evade the human immune system. Successful completion of these aims will improve our understanding of the normal processes of NK cell development with the goal of applying these discoveries toward novel immune-based therapies for cancer.